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Dynamic genetic adaptation of Bacteroides thetaiotaomicron during murine

gut colonization

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Dynamic genetic adaptation of Bacteroides

thetaiotaomicron during murine gut colonization
Graphical abstract Authors
Megan S. Kennedy, Manjing Zhang,
Orlando DeLeon, ..., Phoebe A. Rice,
Joy Bergelson, Eugene B. Chang

Correspondence
echang@medicine.bsd.uchicago.edu

In brief
Kennedy et al. evaluate temporal
dynamics of Bacteroides
thetaiotaomicron (Bt) adaptation to a new
host. Bt metabolic priorities shift over the
course of colonization, culminating in
strong selective pressure to efficiently
consume dietary resources. This work
highlights the importance of considering
temporal dynamics in developing better
microbiome-based therapies.

Highlights
d B. thetaiotaomicron adaptation to the germ-free mouse gut is
a dynamic process

d Early selective pressures are distinct from intermediate and

long-term pressures

d Selective pressure for efficient dietary resource use

dominates in the long term

d Changes in Bt spatial localization parallel changes in

resource use

Kennedy et al., 2023, Cell Reports 42, 113009

August 29, 2023 ª 2023 The Authors.
https://doi.org/10.1016/j.celrep.2023.113009 ll
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Resource
Dynamic genetic adaptation
of Bacteroides thetaiotaomicron
during murine gut colonization
Megan S. Kennedy,1,2,14 Manjing Zhang,3,14 Orlando DeLeon,4 Jacie Bissell,4 Florian Trigodet,4 Karen Lolans,4
Sara Temelkova,4 Katherine T. Carroll,4,12 Aretha Fiebig,5 Adam Deutschbauer,6,7 Ashley M. Sidebottom,8 Joash Lake,9
Chris Henry,10 Phoebe A. Rice,11 Joy Bergelson,3,13 and Eugene B. Chang4,15,*
1Medical Scientist Training Program, Pritzker School of Medicine, The University of Chicago, Chicago, IL, USA
2Department of Ecology & Evolution, The University of Chicago, Chicago, IL, USA
3Committee on Microbiology, The University of Chicago, Chicago, IL, USA
4Department of Medicine, The University of Chicago, Chicago, IL, USA
5Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA
6Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
7Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, USA
8Duchossois Family Institute, Department of Biomedical Sciences, The University of Chicago, Chicago, IL, USA
9Committee on Immunology, The University of Chicago, Chicago, IL, USA
10Mathematics and Computer Science Division, Argonne National Laboratory, Lemont, IL, USA
11Department of Biochemistry & Molecular Biology, The University of Chicago, Chicago, IL, USA
12Present address: Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA
13Present address: Department of Biology, Center for Genomics and Systems Biology, New York University, New York, NY, USA
14These authors contributed equally
15Lead contact

*Correspondence: echang@medicine.bsd.uchicago.edu
https://doi.org/10.1016/j.celrep.2023.113009

SUMMARY

To understand how a bacterium ultimately succeeds or fails in adapting to a new host, it is essential to assess
the temporal dynamics of its fitness over the course of colonization. Here, we introduce a human-derived
commensal organism, Bacteroides thetaiotaomicron (Bt), into the guts of germ-free mice to determine
whether and how the genetic requirements for colonization shift over time. Combining a high-throughput
functional genetics assay and transcriptomics, we find that gene usage changes drastically during the first
days of colonization, shifting from high expression of amino acid biosynthesis genes to broad upregulation
of diverse polysaccharide utilization loci. Within the first week, metabolism becomes centered around utili-
zation of a predominant dietary oligosaccharide, and these changes are largely sustained through 6 weeks
of colonization. Spontaneous mutations in wild-type Bt also evolve around this locus. These findings
highlight the importance of considering temporal colonization dynamics in developing more effective micro-
biome-based therapies.

INTRODUCTION colonization of the mammalian gut but has largely neglected

Rapid adaptation is paramount to the survival of any species un-
dergoing an environmental transition. For microbial taxa, which
are frequently and rapidly dispersed across dramatically different
habitats and microenvironments, processes of local adaptation
may arise as primary determinants of microbial colonization suc-
cess and resulting biogeography.1 The mammalian gut is an envi-
ronment regularly bombarded with a diverse array of exogenous
microorganisms. As such, it represents a biologically and clini-
cally relevant system to explore rapid microbial adaptational
processes.
An emerging body of literature has begun probing the evolu-
tionary and selective dynamics of exogenous microbes during
early-time-point or transient dynamics en route to long-term
persistence. For instance, several studies using transposon
sequencing (TnSeq)-based approaches have evaluated the ge-
netic requirements for colonization across various microbes in
the guts of conventionally raised, gnotobiotic, and germ-free
mice, but have assessed fitness only at a single time point after
introduction.2,3 Wu et al. evaluated TnSeq mutant abundances of
four Bacteroides strains over a 16-day time course but performed
only broad characterization of population-level shifts at intermedi-
ate time points, restricting more rigorous gene-level functional an-
alyses to a single endpoint.4 Several recent evolution studies have
demonstrated the extent to which carbon limitation and metabolic
demands drive the evolutionary trajectories of commensal and

Cell Reports 42, 113009, August 29, 2023 ª 2023 The Authors. 1
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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probiotic strains such as Bacteroides thetaiotaomicron (Bt) or Es-
cherichia coli in the gut.5–8 However, despite dense time-course
sampling, these studies do not identify genes that are important
for fitness specifically at earlier stages of colonization. On shorter
adaptive timescales, transcriptomics analyses of commensal mi-
crobes such as Bt in vivo and in vitro have demonstrated that
gene expression profiles adapt quickly to factors such as diet,9
community membership,10,11 immune activation,12 or spatial
localization within the gut.13 However, no studies have yet compre-
hensively evaluated the temporal transcriptional profile of a
commensal microbe over the course of colonization.
In the following experiments, we introduce a human-derived
commensal organism, Bt, into the guts of germ-free C57Bl/6
mice to determine whether the genetic requirements for coloni-
zation shift over time and, if so, to characterize the biological
functions required for microbial survival at different stages of
colonization and persistence. Use of a germ-free monocoloniza-
tion model allows us to reduce the staggering complexity of the
gut microbial ecosystem into experimentally tractable and
readily interpretable components: here, we rigorously outline
population-level microbial colonization dynamics for a widely
used model organism and the host-microbe interactions that
drive them. Using this germ-free model as a baseline, future
work can evaluate the distinct contributions of microbe-microbe
interactions and other emergent community-level properties to
community assembly and colonization dynamics.
To identify the microbial genes important for fitness in this
context, we combine two complementary unbiased approaches:
transcriptomics (RNA sequencing), which reveals global gene us-
age patterns, and a functional genetics approach (BarSeq3,14 [bar-
coded anatomy resolved by sequencing]) to assess fitness conse-
quences of gene disruptions at a global scale over the course of gut
colonization. We validate these results with both in vivo metabolo-
mics analysis and in vitro microbial growth experiments. Finally, we
evaluate spontaneous evolution of wild-type (WT) Bt in the gut to
survey natural population-level fitness dynamics. Our results indi-
cate that adaptation to the host gut occurs in distinct stages. Dur-
ing the earliest stage of colonization, genes involved in amino acid
and vitamin biosynthesis are upregulated and, in some cases, play
essential roles in survival of Bt. By colonization days 2–4, Bt shifts
toward upregulation of a diverse array of carbohydrate metabolism
genes. This broad survey of available resources continues through
day 7 before ultimately centering on upregulation of a polysaccha-
ride utilization locus (PUL) responsible for the degradation of raffi-
nose-family oligosaccharides (RFOs) rich in the standard chow
diet fed to our mice. This metabolic shift accompanies a change
in Bt localization from the mucus toward the lumen, where the
expression profile remains largely consistent through at least
6 weeks of colonization. Spontaneous mutations in WT Bt also
evolve around the same PUL, highlighting the importance of effi-
cient carbohydrate metabolism for long-term persistence.
These experiments lay the groundwork for future delineation of
shifting selective pressures in various host backgrounds and mi-
crobiome compositions. We expect that these insights into the
temporally dynamic stresses that microbes must overcome to
colonize and persist in the gut will prove invaluable to our under-
standing of microbial adaptation and the development of micro-
biome-based therapies.
2 Cell Reports 42, 113009, August 29, 2023
Resource
RESULTS
Both transcriptional and genetic fitness determinants
shift over the course of Bt colonization and persistence
To evaluate global transcription during colonization, we intro-
duced WT Bt into germ-free (GF) C57Bl/6 mice and collected
cecal contents at days 0.5, 1, 2, 4, 7, 14, and 42 after colonization
(Figure 1A, n = 3–4 mice per time point). A control cohort of GF
mice was gavaged with sterile phosphate-buffered saline, and
in vitro control samples were collected from n = 4 Bt cultures
at mid-log phase in brain heart infusion supplemented medium.
After rRNA and host RNA depletion, the bacterial RNA samples
were sequenced and compared across time points. In parallel,
to assess functional genetic requirements during colonization,
we introduced a rich library of randomly barcoded Tn insertion
(RB-Tn) mutants of Bt into four different cohorts of GF C57Bl/6
mice (n = 3–5 mice/cohort) and collected near-daily fecal sam-
ples (Figure 1A). Amplification and sequencing of the transposon
barcodes reveals the relative abundance of each mutant in the
library at each time point.14 RB-Tn experiments were carried
out according to two slightly different protocols, with adjust-
ments made to optimize experimental logistics and reduce
bottleneck effects (Figure S1 and STAR Methods).
First, we asked whether there are differences in Bt gene
expression at different times after introduction into the mouse
gut. We first performed principal coordinates analysis (PCoA) us-
ing Bray-Curtis dissimilarity on all gene expression data (Fig-
ure 1B). We performed PERMANOVA analysis including both
experimental day and cohort as explanatory factors, and found
that only experimental day significantly contributed to clustering,
explaining 79% of the variation in the dataset (Table S3).
We next performed post hoc analyses to evaluate clustering by
experimental day. We confirmed that the in vitro expression pro-
file of Bt clustered distinctly from all other time points (adjusted
p < 0.05 for all comparisons, Table S3) and excluded in vitro sam-
ples from further analysis. Examining in vivo results, we found that
day 0.5 and day 1 were statistically indistinguishable, as were day
2 and day 4, and day 14 and day 42, but that all other time points
formed significantly distinct clusters (Figure 1B and Table S3).
Therefore, for all further analyses, day 0.5 and day 1 data were
combined (‘‘D0.5/D1’’), day 2 and day 4 data were combined
(‘‘D2/D4’’), and day 14 and day 42 data were combined (‘‘D14/
D42’’). The largest changes in gene expression, in which samples
traverse principal coordinate 1 (PC1), occurred within 2 days after
introduction to the gut. Although day 7 and D14/D42 were statis-
tically distinct, these clusters spanned a smaller and overlapping
range within PCoA space, and therefore reflect relatively minor
changes in global gene expression profile. To further assess
overarching gene expression changes in Bt over 6 weeks of gut
colonization, all genes that met stringent criteria of differential
expression (log false discovery rate [FDR] <
3, |log2fold change
[FC]| > 2, and base mean >50 reads per million [RPM]) at any time
point relative to day 1 were displayed in a heatmap (Figure 1C and
Table S5). Even by these stringent criteria, we identified 489
differentially expressed genes (DEGs), which show a temporal
pattern of relative peaks in expression at different time points.
Consistent with these global changes in gene expression over
time, PCoA ordination using the relative abundance profiles of

Resource
A Figure 1. Bt gene expression and genetic
B D
C riched on D0.5/D1 and expression of 192
the RB-Tn mutant strains within each mouse in our functional ge-
netics experiment reveals that the mutant pool composition shifted
across time in a replicable pattern (Figure 1D) across four indepen-
dent cohorts of this experiment, despite protocol adjustments.
PERMANOVA showed that the data cluster significantly by exper-
imental day (p = 0.0001, R2 = 0.457), with less substantial but sig-
nificant contributions by individual mouse ID (p = 0.0001, R2 =
0.135) and experimental cohort (p = 0.0001, R2 = 0.092). The
largest shifts across PC1 occurred between day 1 and day 7,
with the mutant pool changing less dramatically between day 7
and day 14. Together, these data suggest that different sets of
genes mediate colonization and growth immediately upon gut en-
try and later in the adaptational process.
Amino acid and vitamin biosynthesis are
transcriptionally upregulated and functionally
significant during early colonization of the gut
To gain a more comprehensive understanding of the gene path-
ways expressed during the acute phase of adaptation to the gut,
we performed pairwise comparisons of gene expression across
sequential time points. We identified extensive shifts in the Bt tran-
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fitness determinants shift over the course of
colonization and persistence
(A) Experimental scheme of transcriptomics (top)
and functional genetics (bottom) experiments in
germ-free (GF) mice.
(B) Principal coordinates analysis (PCoA) using
Bray-Curtis dissimilarity on the global gene expres-
sion profile of WT Bt at 0.5, 1, 2, 4, 7, 14, and 42 days
of colonization (n = 4–7 per group).
(C) Heatmap visualizing all genes that were signifi-
cantly differentially expressed at any time point
relative to day 1 (criteria: logFDR <
3, |log2FC| > 2,
base mean >50 RPM). Each column represents an
individual gene (Table S5); data are normalized
across all time points for each gene.
(D) PCoA using Bray-Curtis dissimilarity on the
abundances of RB-Tn mutant strains in fecal sam-
ples, colored by experimental day, shaped by
experimental cohort. Four different cohorts of mice
(n = 3–5 per cohort) are represented.
See also Tables S3 and S5.
scriptome between D0.5/D1 and D2/D4,
with expression of 85 genes relatively en-
genes enriched at D2/D4 (Figure 2A). The
pathways enriched at D0.5/D1 comprised a
largely unique set of genes from those that
dominate the Bt expression profile in vitro
(Table S5). To functionally characterize these
DEGs, we mapped them to the Kyoto Ency-
clopedia of Genes and Genomes (KEGG)
catalog (Figure 2B). Although fewer DEGs
were upregulated at D0.5/D1 compared to
D2/D4, more of these genes mapped to
known KEGG pathways, spanning a variety
of functions, many of which center around
metabolism. In particular, 30 DEGs upregulated at D0.5/D1 map-
ped to amino acid metabolism functions compared to ten amino
acid genes upregulated at D2/D4.
To further explore the metabolic functions characteristic of the
early phase of colonization, we performed gene set enrichment
analysis (GSEA) for all KEGG metabolism modules across D0.5/
D1 and D2/D4 (Figure 2D). Expression of pathways corresponding
to the biosynthesis of many amino acids was enriched specifically
at the D0.5/D1 time point. Expression of genes involved in the
biosynthesis of biotin, a cofactor required for amino acid biosyn-
thesis, was concurrently upregulated at D0.5/D1. These results
are supported by RB-TnSeq analysis: by mapping the significantly
depleted gene mutants (t statistic <
3s) on the first day of the func-
tional genetics assay using KEGG, we identified amino acid meta-
bolism as the KEGG family with the largest number of significantly
depleted mutants (Figure 2C). These genes are identified by gold
stars on the amino acid biosynthesis pathway map in Figure S2,
which illustrates reactions with upregulated gene expression at
D0.5/D1 compared to D2/D4. We observe that biosynthesis of
most amino acids involves multiple reactions that are transcription-
ally enriched at D0.5/D1, or one step that is functionally essential.
Cell Reports 42, 113009, August 29, 2023 3
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A B
C with no KEGG annotations were excluded.
D E
Not only do both our transcriptomics analysis and functional ge-
netics screen support a key role for amino acid biosynthesis early
in colonization, but metabolomic analysis of the cecal contents
corroborates this finding. We measured the levels of specific
amino acids in the cecum of GF mice and compared those to cecal
levels in ex-GF mice at day 1, day 7, and day 14 after colonization
with WT Bt, and found that amino acid levels were generally higher
on day 1 than on day 7 or day 14 (Figure 2E), as shown previously in
an E. coli colonization model.8 This difference was especially pro-
found and statistically significant for amino acids in the glycine-
serine-threonine pathway and for asparagine (Table S4). By
contrast, glutamate and proline reach their highest levels at day
7 before being depleted in the second week.
Although upregulation of amino acid and vitamin biosynthesis at
D0.5/D1 is reminiscent of the stringent response (SR), in which
growth is inhibited under conditions of nutrient limitation to priori-
tize biosynthesis of essential nutrients,15 further analysis suggests
that early colonization is a distinct transcriptional program. Using a
4 Cell Reports 42, 113009, August 29, 2023
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Figure 2. The Bt transcriptome undergoes
dramatic remodeling during the first week af-
ter introduction to the murine gut
(A) Volcano plot of significant transcriptional differ-
ences between day 0.5 (D0.5)/D1 and D2/D4.
Eighty-five genes had significantly increased
expression on D0.5/D1 (red), while 192 had signifi-
cantly increased expression on D2/D4 (blue) (cutoff:
log(FDR-adjusted p value) <
3, |log2FC| > 2,
maximum group mean >50 RPM).
(B) DEGs from (A) colored by KEGG family. Genes
(C) Number of gene mutants significantly depleted in
the RB-Tn experiment (t statistic <
3s) on day 1 of
the experiment, plotted by KEGG family.
(D) GSEA using transcriptomics data for all KEGG
metabolic modules, comparing D0.5/D1 to D2/D4.
Only pathways with significant differential expres-
sion (p < 0.05) are shown.
(E) Abundances of amino acids in the ceca of ex-GF
mice at day 1, day 7, and day 14 of colonization
measured using gas chromatography-mass spec-
trometry (GCMS) and normalized to internal stan-
dards and to respective GF day-0 controls (n = 5
mice for GF day 0, n = 4 for post-colonization sam-
ples). See also Table S5.
list of Bt-specific (p)ppGpp-mediated SR
pathways manually curated from transcrip-
tomic analyses by Schofield et al.,16 we per-
formed GSEA to determine whether SR tran-
scriptional patterns were better represented
at D0.5/D1 or D2/D4. Almost all pathways
expected to be downregulated during SR
were downregulated at D2/D4 rather than
D0.5/D1, but pathways expected to be up-
regulated during SR were generally upregu-
lated at D0.5/D1 (Table S5). Thus, although
some pathway targets of the (p)ppGpp-
mediated SR transcriptional program may
be differentially expressed during gut coloni-
zation, these likely represent a distinct but overlapping response to
the shifting selective pressures of the gut resource environment.
A shift toward enhanced expression of diverse sugar
metabolism genes occurs during the first week of gut
colonization
Of the 192 DEGs upregulated at D2/D4 compared to D0.5/D1, only
50 mapped to known KEGG orthologs (Figure 2B and Table S5).
Among these, we noted specific enrichment of genes involved in
transcription, membrane transport, and genetic information pro-
cessing, which represents Bt’s seven identical insertion sequence
3 (IS3)-family transposases. In the functional genetics experiment,
we found that by day 2, amino acid metabolism had been sur-
passed by carbohydrate metabolism as the KEGG family with
the largest number of depleted gene mutants (Figure 3A). This sug-
gests that amino acid metabolism becomes less functionally
essential after day 1 and that efficient carbohydrate metabolism
becomes more essential.
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A B

C D

E F G

Figure 3. A shift toward greater expression of diverse sugar metabolism genes occurs during the first week of gut colonization
(A) Number of gene mutants significantly depleted from day 1 to day 3 of the RB-Tn experiment (t statistic <
3s), colored by KEGG family.
(B–G) Number of PULs significantly differentially expressed (adjusted p < 0.05) across sequential pairwise comparisons identified via GSEA using transcriptomics data.
Specific PULs differentially expressed across (C) day 0.5 (D0.5)/D1 vs. D2/D4 and (D) D2/D4 vs. D7 identified via GSEA. Predicted PUL substrates are listed on the right.
Mean expression levels (RPM) for all genes in the (E) PUL24, (F) PUL27, and (G) PUL6 operons. Error bars excluded for visual clarity; statistical comparisons done via GSEA.
See also Table S5.

Members of the genus Bacteroides are well known for their ability
to digest a wide variety of polysaccharides.17–19 According to the
CAZy database, Bt possesses 359 glycoside hydrolases, 87
glycosyl transferases, 15 polysaccharide lyases, and 19 carbohy-
drate esterases.20 To further probe carbohydrate utilization, we
performed GSEA on all PULs in the Bt genome in pairwise compar-
isons across sequential time points. Across all time points, 49
unique PULs were significantly differentially expressed. We find
that from D0.5/D1 to D2/D4, expression of 21 PULs was upregu-
lated compared to only six that were downregulated (Figures 3B

Cell Reports 42, 113009, August 29, 2023 5

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and 3C). From D2/D4 to day 7, 12 PULs were upregulated
compared to eight that were downregulated (Figures 3B and 3D),
and from day 7 to D14/D42, more PULs were downregulated
than upregulated (Figure 3B). Collectively, these data show that
over the first week of colonization, Bt increases expression of a
broad array of PULs, but expression of many of these PULs is
reduced as time goes on, perhaps to optimize utilization of avail-
able resources.
To identify specific PULs that may be particularly central to
efficient resource utilization, we searched for PUL genes whose
mean expression across mice increased monotonically over all
time points, and found genes belonging to 21 PULs, including
PUL24 and PUL59, both of which are adjacent to IS3-family
transposases (Table S5). Of these 21 PULs, only PUL24,
PUL27, and PUL6 had expression patterns that were broadly
consistent across all genes in the PUL (Figures 3E and 3F).
These PULs moreover reached substantially higher levels of
expression (1,500–6,000 RPM) than any of the other 18
PULs (200–400 RPM). PUL27 and PUL6 are predicted to
enable degradation of host mucosal glycans, suggesting that
Bt may be foraging for sugars through degradation of the
mucus layer.17 The last gene of PUL24 encodes an a-galacto-
sidase (BT1871), which is predicted to confer the ability to hy-
drolyze the a-1,6 glycosidic linkage in RFOs, a major compo-
nent of the fiber-rich diet fed to our mice, as well as many
standard mouse chows.5 Although this study focuses on under-
standing the shifting genetic determinants of colonization over
time, we recognize that the effect of diet is intrinsic to the re-
sults of any gut microbiome study.
Global Bt gene expression stabilizes after 1 week of
colonization
In contrast to the 277 DEGs identified between D0.5/D1 and
D2/D4, we found only 21 DEGs that met our criteria between
D2/D4 and day 7, and only seven between day 7 and D14/
D42 (Table S5). Of the DEGs between D2/D4 and day 7, the
only one that mapped to any KEGG pathway was in the
PUL24 operon, and similarly, five of the seven DEGs between
day 7 and D14/D42 fell into the PUL24 operon. Thus, we infer
that Bt’s transcriptional profile has largely stabilized within
the first week of colonization and is maintained thereafter,
excepting the continued upregulation of certain PUL genes.
Upregulation of a-galactosidase activity confers a
significant growth advantage to Bt in GF mice fed a
standard RFO-rich diet
Our functional genetics screen confirmed the importance of
PUL24: around 4 days after introduction of the RB-Tn mutant li-
brary into GF mice, the diversity of the community begins to
collapse due to strong positive selection for a small pool of mu-
tants that seemed to have gained fitness from the Tn insertion
(Figure 4A). This could be caused by RB-Tn insertions in regula-
tory elements or by polar effects on downstream genes driven by
readthrough from the antibiotic resistance promoter of the RB-
Tn insertion.3 RB-Tn mutants with insertions in PUL24 were
among the most positively selected mutants at the end of the
2-week experiment, exhibiting growth that exceeded a neutral
expectation that final abundances would simply reflect initial
6 Cell Reports 42, 113009, August 29, 2023
Resource
abundances in the inoculum (Figure 4B). Furthermore, most of
the hyperfit mutants had Tn insertions in one of three operons:
PUL24 (BT1871–1877), PUL39 (BT2851–2860), or BT3130–
3134, all of which encode at least one a-galactosidase, situated
at the tail end of the operon (Figure 4C). At the end of the RB-Tn
selection experiments, the populations were overtaken by mu-
tants carrying Tn insertions upstream of these a-galactosidase
genes (Figure 4D). This was true for all mice across four indepen-
dent experimental cohorts, which were carried out months apart
from one another (Figure 4E).
This RB-Tn pool has previously been assayed in over 300
different conditions including distinct carbon or nitrogen sources
and specific stress conditions.3 The mutants that we identified
as hyperfit in GF mice exhibit a phenotype significantly deviant
from WT in only two conditions: within GF mice and in defined
culture with melibiose—a disaccharide of glucose and galac-
tose, and a breakdown product of the trisaccharide raffinose
(Figure S3A)—as the sole carbon source. In both cases, these
mutants exhibit a growth advantage. Indeed, when we isolated
the most abundant strains from day 14 of the RB-Tn experiment,
we found that their growth rate was significantly higher than WT
when melibiose is the sole carbon source (Figure 5A and
Table S6). In this condition, Bt must hydrolyze the a-1,6 glyco-
sidic bond to harvest and metabolize the monosaccharide
sugars. The competitive advantage of these mutants cannot
be attributed to either of the monosaccharides or to hydrolysis
of a-1,2 glycosidic bonds, as the mutant growth rates on these
carbon substrates (glucose, galactose, and sucrose) are indistin-
guishable from WT (Figure S3B). In raffinose medium, which con-
tains both a-1,2 and a-1,6 linkages, mutants exhibit an attenu-
ated but still significant growth advantage (Figure S3B, p =
3.9e
05, t test, n = 4–6).
Given that release of the monosaccharide sugars of meli-
biose depends on hydrolysis of an a-1,6 glycosidic bond, we
hypothesized that the fitness phenotypes both in mice and
in vitro did in fact depend on overexpression of the a-galacto-
sidase gene downstream of the Tn insertion site. To test the hy-
pothesis that the Tn insertion enhanced expression of down-
stream genes, we grew WT and hyperfit mutants in melibiose
medium and measured the expression of a-galactosidase
genes. For this experiment we used two mutants, one carrying
an insertion in PUL24 and another carrying an insertion in the
BT3130–3134 operon. For both mutants, we found >10-fold
overexpression of a-galactosidase downstream of the insertion
but within the same operon (Figure 5B, BT1871: p < 1e
6, t
test; BT3131: p = 4e
6, n = 3–4/group). Meanwhile, expression
of an a-galactosidase (BT2851) located outside the operons
that carry the insertion was similar between WT and the
mutants.
We then overexpressed BT1871 from a strong, constitutively
active promoter (PrpoD) in WT Bt and observed a 3-fold increase
in log-phase growth rate relative to WT when melibiose was the
sole carbon source (Figure 5A and Table S6). We note that even
this constitutive-expression mutant did not exhibit as much
growth advantage as the RB-Tn mutant strains. It is possible
that the RB-Tn strains may have evolved additional mutations
that further promote growth on melibiose media, although we
identified no new junctions suggesting genomic rearrangements

Resource
A D
B final abundance; red line represents a linear regres-
E
C tions averaged across mice for each experimental run.
and no changes in coverage suggesting duplications in whole-
genome short-read sequencing of RB-Tn isolates. Alternatively,
the promoter used in front of the erythromycin resistance
cassette in the RB-Tn mutants may simply be stronger than
the PrpoD promoter we used here.
Metabolomic measurements of a carbohydrate panel confirm
that raffinose and its constituent sugars are among the most
abundant carbohydrate substrates within the ceca of GF mice
fed a standard chow (Figure 5C). Although mammals can cata-
lyze hydrolysis at the a-1,2 linkages in raffinose and sucrose,
the a-1,6 linkages in raffinose and melibiose can only be hydro-
lyzed by gut microbes (Figure S3A).22 Accordingly, raffinose and
melibiose build up in the cecum of our GF mice at high concen-
trations until Bt is introduced. After 7 days, during which time Bt
has initiated overexpression of a-galactosidase genes, these
sugars are depleted (Figure 5D, p = 0.02857, Wilcoxon test,
n = 4; Figure S3C, p = 0.006193, t test, n = 4). In contrast, sucrose
is consumed by the host and no significant changes are
observed in sucrose concentration following Bt colonization
(Figure S3D).
Together, we find that when mice are fed standard high-fiber
chow, the high concentration of RFOs accumulating in the
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OPEN ACCESS
Figure 4. Colonization of a complex Bt
mutant library within GF mice selects for dis-
ruptions upstream of a-galactosidase genes
A–D) (A, B, and D) Data from a single experimental run
(October). (A) Shannon diversity of the RB-Tn mutant
pool in mice over time. Each point represents the RB-
Tn pool within a single mouse on the indicated day
(**p < 0.01, t test, n = 5/time point). (B) Initial (inoculum,
day 0) vs. final (day 14) relative abundance of RB-Tn
mutant strains. Each point represents the summed
relative abundance of all mutant strains that mapped
to given gene, averaged across mice. Dashed gray
line designates a 1:1 relationship between starting and
sion best-fit line generated from the log-transformed
data (p < 2e
16, R2 = 0.6461; STAR Methods). (C)
Organization of PUL 24 (BT1871–1877), an unnamed
PUL containing BT3130–3134, and PUL 39 (BT2851–
2860). Predicted a-galactosidases are colored yellow.
(D) Relative abundance of RB-Tn mutant strains over
time. Each line represents the summed relative
abundance of all mutant strains that mapped to a
given gene, averaged across mice. Top 15 most
abundant gene mutants at day 14 are colored by the
PUL to which they mapped; all others are black.
(E) Day-14 relative abundance of gene mutants
mapping to operons that encode a-galactosidase
functions (yellow), known PULs, or other gene func-
lower gastrointestinal (GI) tract creates
an environment that strongly selects for
Bt strains that can make efficient use of
these sugars through increased expres-
sion of a-galactosidases. We infer that
efficient carbohydrate metabolism—par-
ticularly for abundant dietary fibers—is a
major determinant of population-level se-
lective dynamics during the persistence phase of Bt engraft-
ment within the gut.
Changes in resource-use strategies coincide with shifts
in Bt localization from the mucus toward the lumen
We next sought to determine whether Bt localization changes
over the course of colonization. To assess Bt localization at
days 1, 2, 4, 7, and 42, we labeled fixed colonic cross-sections
with antibodies to MUC2, the predominant colonic mucin,23
and the DNA stain 40 ,6-diamidino-2-phenylindole (DAPI), and
imaged them with a laser-scanning confocal microscope. We
then quantified the average epithelial proximity of Bt by
measuring the distance from the epithelial surface along
perpendicular tracts to the nearest bacterial cell (STAR
Methods).
At days 1 and 2, Bt was deeply embedded within a largely
unstructured mucus layer, often directly adjacent to the epithe-
lium (Figures 6A and 6B). By day 4, an observable but thin and
patchy inner mucus layer began to form (Figure 6C), which
expanded in thickness by day 7 (Figure 6D). This layer still al-
lowed substantial penetration by Bt, although mean distance
from Bt to the epithelium was significantly increased compared
Cell Reports 42, 113009, August 29, 2023 7
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A B
C D
to day 1 (Figure 6F, p = 0.037, t test, n = 4/group). At day 42, a
defined inner mucus layer covered the epithelial surface of
each cross-section, effectively excluding Bt, which localized
primarily to the shedding outer mucus layer and luminal space
(Figures 6E and 6F, p = 0.024, t test, n = 3–4/group). Thus, as
the impermeable mucus layer forms, we identify a shift in Bt
spatial localization from deeply embedded in the mucus layer
early in colonization toward the luminal space by day 7 and
onward.
Our RNA-sequencing data indicated that Bt undergoes a meta-
bolic shift around day 7 of colonization, switching from broadly
upregulated PUL metabolism, including many loci predicted to
encode mucosal glycans, to a metabolic profile strongly centered
around dietary RFO consumption. Here, we find that Bt spatial
localization similarly shifts around day 7 of colonization from the
mucus into the lumen. We have therefore identified a loose corre-
lation between the timing of shifts in Bt resource-use strategies
and its spatial localization with respect to the mucus layer and
luminal space.
8 Cell Reports 42, 113009, August 29, 2023
Resource
Figure 5. Upregulation of a-galactosidase
activity confers a significant growth advan-
tage to Bt in GF mice fed a standard RFO-
rich diet
(A) Log-phase growth rates (k) of the most abundant
RB-Tn mutant strains isolated from mice and grown
in melibiose minimal medium. Strains with asterisks
carry other mutations in addition to the transposon
insertion. The WT + BT1871OE strain carries a
plasmid copy of BT1871 expressed from the rpoD
promoter that was integrated into the WT genome at
the attN1 site. All mutant strains grew significantly
faster than WT (p < 0.0001).
(B) Normalized abundance of a-galactosidase
mRNA (RPM) measured in WT and hyperfit RB-Tn
mutants isolated from mice and grown in Varel-
Bryant defined medium with 20 mM melibiose as
the sole carbon substrate.21
(C) Abundance of various sugars in the GF mouse
cecum as measured using GCMS and normalized to
internal standards.
(D) Abundance of raffinose in the standard chow fed
to GF mice, within GF ceca before colonization, or 1,
7, or 14 days post colonization.
*p < 0.05; **p < 0.01; ***p < 0.0001, ****p < 0.00001.
See also Table S6.
Strong selection for efficient RFO
metabolism leads to emergence of
spontaneous Bt mutants with
duplicates of the BT1871 locus
through an IS3-family transposable
element
The transposon mutants in our functional
genetics experiment were able to gain
10-fold increase in a-galactosidase exp-
ression due to readthrough from an ext-
remely strong, synthetic promoter. We
wondered whether the selective pressure
for elevated a-galactosidase activity to uti-
lize the a-1,6-linked sugars abundant in the diet would drive evo-
lution of a spontaneous mutant with enhanced a-galactosidase
activity. One week after the initial colonization of GF mice with
WT Bt in an ongoing transcriptomics experiment, three mice
from two cages were separated into individual cages for 6 weeks
of observation, a length of time predicted to be sufficient for
spontaneous mutations to arise and stabilize.7 At the end of
6 weeks, we performed shotgun sequencing on fecal material
from all three mice. Assembly of the short-read sequences re-
vealed that there was at least 23 coverage of the BT1871 locus
for all samples (Figure 7A). A dip to 13 coverage in the middle of
this region mapped to an IS3-family transposase, of which there
are six other identical copies in the genome; hence, the corre-
sponding coverage was diluted among the different copies.
BreSeq analysis identified three new junctions in the Bt genome
indicative of genome rearrangements (Figure 7B). Notably, sam-
ples from mouse A and mouse C, which were co-housed during
the first week of the experiment, shared an identical junction,
suggesting that this mutation arose in Bt within the first week

Resource
A B C Figure 6. Bt localization shifts from the
D E F
of colonization and was transmitted across mice. Samples from
mouse B and mouse C each possessed unique junctions, indi-
cating that these junctions arose independently. In all, three in-
dependent junctions spontaneously evolved in this experiment,
and all three junctions were formed between either BT1872 or
BT1873 and a locus downstream of BT1871, which encodes
for rRNAs, tRNAs, and a ribosome recycling factor. All three
new junctions occupied a significant portion of the sequencing
reads (60%–73%), suggesting that the mutants carrying these
junctions were competitively dominant in the gut environment.
IS3-family transposases are known to act by a copy-and-
paste mechanism.24 We wondered whether this transposase,
which we had identified as significantly upregulated from D0.5/
D1 to D2/D4 (Table S5), could have created multiple copies of
the BT1871 locus. We isolated individual strains likely to contain
these mutations by plating bulk fecal material and then selecting
a single strain from each mouse that exhibited a growth advan-
tage compared to WT on melibiose medium (Figure 7C). We
then performed long-read sequencing of each strain with
MinION. In one of these isolates, we identified a tandem repeat
of the BT1871 locus (Figure 7D), which places the original copy
of BT1871 downstream of a strong rRNA promoter. Interestingly,
the six identical copies of this IS3-family transposase (NCBI:
60924995) are often found adjacent to PULs in the WT Bt
genome. This suggests that these transposable elements confer
a mechanism to modulate gene expression that may be advan-
tageous in adapting to new metabolic landscapes.
Interactions with the host are relatively consistent
throughout colonization
Although the host response to monocolonization is not the focus of
this paper and has been thoroughly cataloged elsewhere,25–28 we
assessed levels of cecal immunoglobulin A (IgA) and serum IgG, as
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OPEN ACCESS
mucus toward the luminal space over coloni-
zation
(A–E) Representative cross-sections of distal mouse
colon fixed in Carnoy’s solution at (A) day 1 (D1), (B)
D2, (C) D4, (D) D7, and (E) D42 of colonization by Bt.
Blue, DAPI staining in the epithelium; red, DAPI
staining in the lumen of gut, including bacteria,
debris, and shed host nuclei; green, antibody
staining for MUC2. Scale bars, 10 mm.
(F) Mean Bt epithelial proximity. n = 3–4/group, t test,
*p < 0.05.
well as several pro-inflammatory cytokines
from colonic mucosal scrapings. We identi-
fied no significant or consistent trends in
any of these metrics (Figure S4) Previous
work has outlined how IgA binding to
commensal bacteria such as Bt may impact
gene expression, in some cases affecting di-
etary polysaccharide usage and microbial
localization.27,29–31 We could not recapitu-
late these specific changes in our Bt gene
expression time course. This may reflect dif-
ferences in IgA assay protocols,29 or it may suggest that Bt’s
response to IgA binding is sensitive to the specific mouse chow
composition and availability of dietary resources.
We then performed targeted GSEA on expression of Bt’s
capsular polysaccharide (CPS) loci, which encode outer mem-
brane proteins that mediate interaction between Bacteroides
genera and the host immune system32 and have been shown
previously to play a critical role in Bacteroides gut coloniza-
tion.2,33,34 We found that CPS loci 1 and 3, and to a lesser extent
2, 4, and 7, were highly expressed early in colonization and then
downregulated, whereas CPS5 and CPS6 had increasing rela-
tive expression from day 7 onward (Figures S5A–S5D). Only
CPS4 mutants showed severe fitness defects during the early
days of the functional genetics assay, in line with previous re-
ports (Figure S5E).2 These data indicate that Bt modifies its outer
membrane upon entry into the gut and over the course of persis-
tence, which may reflect adaptation to the host immune system,
perhaps in parallel to shifts in localization.
DISCUSSION
Microbial adaptation during colonization is a dynamic
process
Bacteroides species are dominant and prevalent in the guts of
many human populations.35,36 Instrumental to their success is
their ability not only to tolerate stress but to quickly adapt in order
to grow under a range of conditions.37,38 Although Bt has been
used as a model organism to understand the genetic drivers of
gut colonization for commensal organisms,2,4,39 a single repre-
sentative time point has been assessed in most previous inves-
tigations, which fail to consider the dynamism of the adaptational
process. For example, whereas previous work based on in vivo
Bt gene expression at a single time point characterized only
Cell Reports 42, 113009, August 29, 2023 9
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A Figure 7. Under strong selective pressure, Bt
B
C D
one metabolic phase that prioritized carbohydrate transport and
metabolism,9 our time-course data suggest that Bt undergoes a
temporally dynamic adaptational program. We show that Bt pri-
oritizes biosynthesis of amino acids and other essential com-
pounds early in colonization, before ramping up expression of
a diverse array of PULs and ultimately centering metabolism
around degradation of a single abundant dietary carbohydrate.
This may reflect a transition from an initial stress response during
establishment in the gut to a more growth-focused strategy that
surveys and optimizes utilization of available environmental re-
sources. We find that the set of genes required for Bt to initially
establish colonization in the gut are distinct from those required
to persist (Figure 1). The results presented here suggest that
because of the rapidly shifting adaptational profile exhibited by
microbial populations over the course of colonization, investiga-
tors must take care in selecting the appropriate time points to
address their experimental questions.
10 Cell Reports 42, 113009, August 29, 2023
Resource
duplicates the BT1871 locus with the help of a
transposable element
(A) Shotgun sequencing of bulk fecal sample from
three mice inoculated with WT Bt shows at least 23
coverage of the BT1871 locus.
(B) New Bt genomic junctions identified via shotgun
sequencing of bulk fecal material from each mouse.
(C) Log-phase growth rates (k) of three mutant
strains isolated from feces of mice colonized with
WT Bt for 6 weeks compared to growth rate of the
ancestral WT Bt in defined medium supplemented
with melibiose (n = 4 technical replicates per strain).
p < 0.05.
(D) Long-read MinION sequencing of an abundant
mutant in mouse C (MZ65) reveals the duplication of
a long segment including the BT1871 gene, which
generates a tandem repeat of the locus. Each gray
bar represents a single long read, which together
span the region containing the tandem repeat.
Early colonization is a distinct
adaptational phase
The process of colonizing a new host pre-
sents a diverse array of environmental
changes that require physiological adapta-
tion. The largest transcriptomic changes
that we observed across 6 weeks of Bt
colonization occurred in the first week (Fig-
ure 1B), as did the largest shifts in RB-Tn
mutant abundances (Figure 1C). Our find-
ings indicate that Bt upregulates a broad
swath of metabolic processes upon arrival
in the lower GI tract during the earliest
stage of colonization (Figure 2B). Work
from Watson et al. examining the qualities
of microbes that successfully colonize the
gut after fecal microbial transplant sug-
gests that microbes that can be metaboli-
cally ‘‘self-sufficient’’ may be better suited
to engraft and persist in a new host envi-
ronment.40 We show that, similarly, Bt ca-
sts a wide metabolic net upon gut entry, upregulating carbohy-
drate metabolism, energy metabolism, vitamin and cofactor
biosynthesis, and especially amino acid biosynthesis. We like-
wise see that Bt upregulates a diverse array of PULs during early
colonization (Figure 3). This may better enable Bt to survive con-
ditions of resource scarcity until a more energetically efficient
transcriptional program can be fine-tuned to the particulars of
its new resource landscape.
Selection for efficient carbohydrate metabolism drives
long-term persistence
During the later stages of colonization and persistence, Bt re-
centers its metabolism around carbohydrate utilization. These
results add to a growing body of work identifying metabolic ne-
cessity in general, and carbohydrate metabolism in particular, as
powerful drivers of microbial fitness and evolution in the gut. For
instance, one study showed that E. coli evolves mutations to

Resource
modulate carbohydrate utilization depending on dietary condi-
tions,6 while another identified selection for mutations in either
amino acid or carbohydrate metabolism depending on the
competitive context.8 Research using Bt has demonstrated
rapid evolution of divergent metabolic strategies depending on
mouse diet.7 One study even showed how microbial competitive
fitness in the mouse gut can be promoted by engineering a
unique PUL into Bt and introducing the strain in combination
with its associated carbohydrate substrate.41 Although microbes
mediate competition in a number of ways ranging from direct
attack to occupation of physical space to indirect interactions
via the host,42 these findings collectively suggest that competi-
tion for scarce metabolic resources is a primary evolutionary
battleground within and among microbial species in the gut
ecosystem.
In these experiments, we identified strong selective pressure
for improved utilization of a specific carbohydrate resource.
However, previous work shows that genetic drift and transmis-
sion of microbes between hosts can significantly impact the evo-
lution and spread of mutant populations.5 Therefore, it is impor-
tant to consider how transmission between hosts may have
impacted our results. In our RB-TnSeq experiments, mice were
co-housed, facilitating intra-cage transmission. This may have
artificially inflated the replicability of our results within cages,
although we still saw near-identical trends emerge across four
independent experiments and eight independent cages.
Changes in microbial localization and metabolic gene
expression profile correlate
We found that changes in Bt resource use over the course of
colonization paralleled shifts in its localization from the inner
mucus layer toward the luminal space (Figure 6). This aligns
with previous work showing that the mucus of GF mice is perme-
able to bacteria and takes up to 6 weeks of conventionalization
to fully mature into the impermeable protective state that ex-
cludes Bt and most other gut commensals.43,44 Although we
provide only correlational evidence here and causal experimen-
tation is beyond the scope of this work, we hypothesize that the
local microenvironment may dictate the selective pressures that
ultimately drive Bt gene expression and evolution: Bt is known to
use mucosal glycans only as a carbohydrate resource of last
resort.45 However, Bt that gets trapped in the permeable imma-
ture mucus layer early in colonization may not have direct access
to luminal dietary resources. Under these resource-limited con-
ditions, the population of mucus-embedded Bt may upregulate
various PULs for consumption of mucosal glycans, as has
been demonstrated in other in vivo mouse studies of Bt under
conditions of dietary resource deficiencies.9,45–48 As the mucus
layer matures and Bt gradually moves into the lumen, its
metabolism shifts from broad mucosal glycan foraging while
embedded (D2/D4) toward hyperutilization of a single abundant
dietary resource while excluded (days 7–42).
Although more rigorous follow-up is needed to establish a
causal relationship between localization and resource use, the
correlation reported here provides preliminary support for the
notion that the spatial localization and metabolic profile of Bt
are deeply intertwined, flexible, and highly responsive to the spe-
cific balance of selective pressures such as resource availability
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and energetic efficiency. These factors will doubtless be heavily
impacted by the presence of additional community members.
Nevertheless, we conclude that resource-use profiling and local-
ization may emerge as particularly informative descriptors of
available niche space in the gut.
IS3 transposable elements: A novel mechanism to
modulate expression of specific CAZymes?
In GF mice fed a standard RFO-rich diet, the selective pressure
for mutants with increased a-galactosidase activity is intense.
Mutations in the BT1871 locus were consistently positively
selected when WT Bt evolved in mice for 6 weeks. In all three
mutant populations, the downstream side of the duplicated re-
gion ends midway through a ribosomal gene, meaning that there
are no known transcriptional terminators between the ribosomal
promoter and the a-galactosidase gene, BT1871. It is likely that
these mutants increased a-galactosidase activity due to read-
through from the strong ribosomal promoter in the upstream
copy of the locus. Although increased a-galactosidase expres-
sion could be simply due to the presence of two copies of
BT1871, the growth phenotype that these mutants exhibit, in
which log-phase growth is five times faster than in WT, is on
par with that of the RB-Tn mutants, for which a-galactosidase
expression was more than ten times greater than in WT.
In Bt, identical copies of this IS3 transposable element occur in
seven locations, often adjacent to PULs and almost always
paired with a ribosomal gene. These genes were upregulated
at D2/D4 of our transcriptomics experiment, shortly before we
saw extensive upregulation of PUL24 genes. It is well known in
both mammals and prokaryotes that transposable elements
can modulate the expression of nearby and distant genes.49
Transposable elements may be selected for because the genetic
‘‘cargo’’ that they shuttle along (such as an antibiotic cassette) is
beneficial to the organism or because the position where the
insertion occurs results in some downstream effect that is bene-
ficial to fitness. Both reasons may contribute to the selection of
the tandem duplication of the BT1871 locus. Future studies will
be necessary to investigate whether other IS3 elements in Bt
perform similar functions in modulating expression of nearby
genes and whether such a mechanism can be found in other
families of bacteria.
Limitations of the study
One limitation of this study is that many microbial genes are
poorly annotated. At most of our transcriptomic time points,
only a minority of the DEGs have known annotations, but those
unannotated genes may still serve meaningful adaptive func-
tions. These genes (Table S5B) would be excellent candidates
for continued functional characterization through controlled
in vitro and in vivo experiments. We also note the key limitation
that this study was performed in a highly simplified monocoloni-
zation model. While these results provide evidence of general
eco-evolutionary principles and highlight important consider-
ations for understanding microbial GI colonization more bro-
adly—e.g., the dynamism of the adaptational process and the
importance of choosing time points carefully, the remarkable ge-
netic mutability of bacteria, and the feedback cycles between a
microbe and its resource environment—the specific results
Cell Reports 42, 113009, August 29, 2023 11
 ll
OPEN ACCESS
presented here may not directly apply to scenarios with more
complex microbial communities. However, by eventually
comparing these results to those from more complex commu-
nities, this dataset can provide even greater insight into how
host-microbe interactions and inter- and intraspecific microbial
interactions each contribute to the adaptational process.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITY
B Lead contact
B Materials availability
B Data and code availability
d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DE-
TAILS
B Animals
B Bacterial strains and growth conditions
d METHOD DETAILS
B In vivo transcriptomic experiments
B In vivo genome-wide mutant fitness assays
B Isolation of mutants from fecal matter
B Host-associated evolution of spontaneous Bt mutants
B Isolate genome sequencing, assembly, and polishing
B Metabolite extraction from cecal material
B Metabolite analysis using GC-EI-MS and methoxy-
amine and TMS derivatization
B RT-qPCR
B ELISA
B Histological procedures
B Immunostaining and imaging
d QUANTIFICATION AND STATISTICAL ANALYSIS
B PCoA, differential expression analysis, and Gene Set
Enrichment Analysis (GSEA) on transcriptomic data
B Pipeline for measuring relative abundance and fitness
scores of RB-Tn mutants
B Metabolomics statistics
B Bacterial growth rate statistics
B Genome mapping and coverage visualization
B BT1871 copy number
B Image quantification
SUPPLEMENTAL INFORMATION
Supplemental information can be found online at https://doi.org/10.1016/j.
celrep.2023.113009.
ACKNOWLEDGMENTS
We thank Hualan Liu for invaluable experimental help with the RB-TnSeq li-
braries. We thank David Hershey and A. Murat Eren for helpful scientific input.
We also thank the Chang lab members for scientific support received. The
graphical abstract was created using BioRender. This work was performed
with support from NIH T32DK007074 (M.Z., M.S.K.), NIH RC2DK122394
(E.B.C.), NIH T32GM007281 (M.S.K.), and the Host-Microbe and Tissue and
Cell Engineering cores of the UChicago DDRCC, Center for Interdisciplinary
Study of Inflammatory Intestinal Disorders (C-IID) (NIDDK P30 DK042086).
12 Cell Reports 42, 113009, August 29, 2023
Resource
AUTHOR CONTRIBUTIONS
Conceptualization, M.S.K., M.Z., E.B.C., and J.B.; methodology, M.S.K., M.Z.,
and E.B.C.; formal analysis, M.S.K., M.Z., O.D., F.T., and A.M.S.; investigation,
M.S.K., M.Z., J.B., K.L., S.T., K.T.C., A.M.S., and J.L.; resources, A.D.; data
curation, M.S.K. and M.Z.; writing – original draft, M.S.K. and M.Z.; writing –
review & editing, M.S.K., M.Z., E.B.C., A.F., J.B., C.H., and P.A.R.; visualiza-
tion, M.S.K., M.Z., O.D., and F.T.; funding acquisition, E.B.C.
DECLARATION OF INTERESTS
The authors declare no competing interests.
INCLUSION AND DIVERSITY
We support inclusive, diverse, and equitable conduct of research.
Received: May 8, 2022
Revised: May 17, 2023
Accepted: August 3, 2023
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Resource OPEN ACCESS

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Goat polyclonal anti-IgA Southern Biotech Cat#OB1040-01
Goat anti-mouse IgA:HRP BioRad Cat#STAR137P
Rabbit polyclonal anti-mouse Muc2 Santa Cruz Biotechnology Cat#sc-15334
Alexa Fluor 488 goat anti-rabbit IgG Invitrogen Cat#A-11008
Bacterial and Virus Strains
Bacteroides thetaiotaomicron VPI-5482 Gift from Deutschbauer lab AMD595
wildtype
RB-Tn mutant isolated from monocolonized Gift from Deutschbauer lab AMD595-derived RB-Tn library3
mice with barcoded transposon insertion in
BT1876
RB-Tn mutant isolated from monocolonized This work Tn_BT1874
mice with barcoded transposon insertion in
BT1874
RB-Tn mutant isolated from monocolonized This work Tn_BT1876
mice with barcoded transposon insertion in
BT1876
RB-Tn mutant isolated from monocolonized This work Tn_BT1872-83
mice with barcoded transposon insertion in
the intergenic region between BT1872 and
BT1873
RB-Tn mutant isolated from monocolonized This work Tn_BT1872
mice with barcoded transposon insertion in
BT1872
RB-Tn mutant isolated from monocolonized This work Tn_BT1874/BT3132
mice with barcoded transposon insertion in
BT1874 and BT3132
RB-Tn mutant isolated from monocolonized This work Tn_BT3133
mice with barcoded transposon insertion in
BT3133
WT Bt with a copy of BT1871 integrated into This work WT + BT1871OE
the Bt genome at the attN1 site under the
BT1311 (rpoD) constitutive promoter.
Bt mutant isolated from feces after 6-week This work MZ65
monoassociation of Bt; carries a tandem
repeat of the BT1871 locus (see Figure 7).
Chemicals, Peptides, and Recombinant Proteins
Brain Heart Infusion Broth Fisher Cat#DF0037178
Agar Fisher Cat#DF0145-17-0
Hemin Fisher Cat#51280-5G
Glucose Sigma Cat#158968-500G
Galactose Sigma Cat#G0750-500G
Sucrose Fisher Cat#AAJ64270A1
Raffinose Fisher Cat#AC195670250
Melibiose Sigma Cat#M5500-100G
Erythromycin Fisher Cat#AC227330050
Phosphate Buffered Saline Sigma Cat#P3813-10PAK
Glycerol Fisher Cat#BP2291
(Continued on next page)

Cell Reports 42, 113009, August 29, 2023 15

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Ambion TRIzol Reagent Fisher Cat#15-596-018
Chloroform Fisher Cat#AC390760010
Isopropanol Fisher Cat#BP2618500
Ethanol Fisher Cat#BP2818500
DNAse Fisher Cat#18068015
Tris Fisher Cat#BP152-10
EDTA Fisher Cat#PR-V4231
NaCl Fisher Cat#S640-10
SDS Sigma Cat#L3771-25G
Phenol:Chloroform:Isoamyl Alcohol Fisher Cat#AM9732
Proteinase K Sigma Cat#3115852001
HCl Sigma Cat#A144-212
Methanol Fisher Cat#A452SK-4
Methoxyamine Sigma Cat#226904
Pyridine Sigma Cat#270970
Derivatizing reagent (BSTFA +1% TMCS) Sigma Cat#B-023
Ethyl acetate Sigma Cat#650528
ELISA diluent R&D Systems Cat#DY995
Acetic acid Fisher Cat#A38-500
Xylene Fisher Cat#X3S-4
Lysozyme Fisher Cat#89833
Sodium citrate Fisher Cat#S279-500
DAPI Sigma Cat#D8417-5MG
ProLong Gold Anti-Fade mounting medium Fisher Cat#P10144
iTaq Universal SYBR Green Supermix BioRad Cat#1725124
Critical Commercial Assays
DNeasy PowerSoil Kit. Qiagen Cat#47016
Rapid Barcoding Kit Oxford Nanopore Technologies Cat#SQK-RBK004
Transcriptor First Strand cDNA Synthesis Roche Cat#35081963001
Kit
Invitrogen IgG (Total) Mouse Uncoated Invitrogen Cat#88-50400-88
ELISA Kit
Deposited Data
RNAseq raw data from Bt at different time This work https://www.ncbi.nlm.nih.gov/sra
points (accession: PRJNA797447)
Shotgun sequencing raw data from This work https://www.ncbi.nlm.nih.gov/sra
evolution experiment fecal samples (accession: PRJNA797447)
Long-read sequencing raw data from This work https://www.ncbi.nlm.nih.gov/sra
isolate MZ65 (accession: PRJNA797447)
Experimental Models: Organisms/Strains
Mouse: germ-free wild-type C57BL/6J University of Chicago N/A
Gnotobiotic Core Facility
Oligonucleotides
Custom Ribo-Zero Plus Probes SeqCenter See Table S1A
qPCR primers Miyoshi et al., 201750 See Table S1B
Software and Algorithms
bcl2fastq Illumina Version 2.20.0.422
MinKNOW Oxford Nanopore Technologies Version 4.3.4
Guppy Oxford Nanopore Technologies Version 5.0.11
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16 Cell Reports 42, 113009, August 29, 2023

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Flye https://github.com/fenderglass/Flye Version 2.651
Pilon https://github.com/broadinstitute/pilon Version 1.2352
MassHunter Quantitative Agilent Technologies Version B.10
Analysis software
R software https://www.r-project.org/ Version 4.2.153
vegan R software https://cran.r-project.org/web/packages/ Version 2.6–454
vegan/index.html
pairwiseAdonis R software https://github.com/pmartinezarbizu/ Version 0.4
pairwiseAdonis
DESeq2 R software https://bioconductor.org/packages/ Version 1.36.055
release/bioc/html/DESeq2.html
anvi’o https://anvio.org/ Version 7.156
fgsea R package https://bioconductor.org/packages/ Version 3.1757
release/bioc/html/fgsea.html
Graphpad Prism https://www.graphpad.com/features Version 9
Prodigal https://github.com/hyattpd/Prodigal Version 2.6.358
bowtie2 https://bowtie-bio.sourceforge.net/ Version v2.3.5.159
bowtie2/index.shtml
samtools http://www.htslib.org/ Version 1.1160
gggenes https://github.com/wilkox/gggenes/tree/ Version 0.4.1
master
blastn https://blast.ncbi.nlm.nih.gov/Blast.cgi Version 2.5.0
minimap2 https://github.com/lh3/minimap2 Version 2.1761
IGV https://igv.org/ Version 2.11.162
LAS_X Leica Leica N/A
Other
Standard mouse chow LabDiets 5K67
Anaerobic chamber Coy Laboratory Products N/A
GENSYS 40-Vis spectrophotometer Thermo Scientific N/A
Mini-BeadBeater-96 BioSpec Products N/A
BioAnalyzer Agilent N/A
NextSeq2000 Illumina N/A
IsoCage P Bioexclusion cages Tecniplast N/A
Agencourt AMPure XP beads Beckman Coulter Cat#A63882
Flow Cell (R9.4.1) Oxford Nanopore Technologies Cat#FLO-MIN106D
MinION Oxford Nanopore Technologies N/A
Beadruptor tubes Fisher Cat#15-340-154
Bead Mill 24 Homogenizer Fisher Cat#15-340-163
Mass spectrometry autosampler vial Microliter Cat#09-1200
Biotage SPE Dry 96 Dual Biotage Cat#3579M
Thermomixer C Eppendorf Cat#2231001005
Agilent 7890A GC system Agilent N/A
Agilent 5975C MS detector Agilent N/A
HP-5MSUI column Agilent Cat#19091S-433UI
CFX384 Real-Time System BioRad N/A
Leica SP8 laser scanning confocal Leica N/A
microscope

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RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources and reagents may be directed to and will be fulfilled by the lead contact, Eugene
Chang (echang@medicine.bsd.uchicago.edu).

Materials availability
Bacterial strains obtained in this study will be made available upon request addressed to the lead contact.

Data and code availability

d All sequencing data, including DNA and RNA datasets, have been deposited at NCBI Sequence Read Archive and are publicly
available as of the date of publication. Accession numbers are listed in the key resources table.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Animals
Female 8-12 week-old C57Bl/6J germ-free mice were bred and maintained in plastic gnotobiotic isolators or bioexclusion racks
within the University of Chicago Gnotobiotic Core Facility and fed ad libitum autoclaved standard chow diet (LabDiets 5K67). Litter-
mates were randomly assigned to treatment groups. All murine experimental procedures were institutionally approved.

Bacterial strains and growth conditions

The bacterial strains used in this study, including the RB-TnSeq library, are listed in the Key Resources Table. Bacteroides thetaio-
taomicron VPI-5482 [AMD595], and all its derivatives were cultured anaerobically at 37 C in liquid Brain Heart Infusion Supplemented
(BHI-S) medium or defined Varel-Bryant medium as described in Liu et al.,3 and Varel and Bryant.21 Varel-Bryant medium with no
carbon source was supplemented with glucose, galactose, sucrose, raffinose, or melibiose to a final concentration of 20 mM. An
anaerobic chamber (Coy Laboratory Products) filled from tanks containing 10% CO2, 7.5% H2, and 82.5% N2 was used for all anaer-
obic microbiology procedures, with working conditions near 2–3% H2.
For growth rate measurements, colonies of Bt were inoculated into 3 mL of BHI-S in plastic culture tubes and grown overnight at
37 C, for a total of 6 biological replicates. Immediately before inoculating with the cells, sealed Hungate tubes containing 10 mL of VB
medium and a 20 mL headspace were inoculated via syringe with autoclaved sugar solution and hemin solution for a final
concentration of 20 mM sugar and 5 mg/mL hemin. Overnight cultures were diluted to OD600 = 1, and 100 mL of the diluted culture
was inoculated into the prepared media tubes via syringe. Anaerobically sealed cultures were grown outside of the chamber in a 37 C
incubator with no shaking. Every 45 min, the cultures were taken from the incubator, cells resuspended by shaking, and their OD600
readings measured by a GENSYS 40-Vis spectrophotometer.

METHOD DETAILS

In vivo transcriptomic experiments

Mice were inoculated with a single dose of wildtype Bt VPI-5482 at 106 - 108 CFU/200 mL, and were then sacrificed at 0.5, 1, 2, 4, 7, 14, and
42 days post-colonization. Luminal contents of the mice cecum were immediately snap-frozen in liquid N2 and stored at
80 C. About
50 mg of the contents were transferred into 2 mL screw-cap tubes, followed by the addition of 1 mL TRIzol reagent for the isolation of
RNA. The samples were homogenized by beadbeating with 0.1 mm glass beads in a Mini-BeadBeater-96 for 2 min. Total RNA isolation
and purification were performed using the TRI reagent protocol and quality checked by BioAnalyzer. All library preparation and sequencing
work was performed by SeqCenter (Pittsburgh, PA). Initial DNAse treatment is performed with Invitrogen DNAse (RNAse free). Library prep-
aration is performed using Illumina’s Stranded Total RNA Prep Ligation with Ribo-Zero plus. Custom Ribo-Zero probes were designed for
Bt and supplemented alongside the standard probe set. Custom probe sequences can be found in Table S1. Sequencing was performed
on a NextSeq2000 giving 2x50bp reads. Post sequencing, bcl2fastq (v2.20.0.422) was used to demultiplex and trim adaptors.

In vivo genome-wide mutant fitness assays

Four individual cohorts of mice were used, indicated by the month of the experiment. For each cohort, mice were housed in cages of
2–3 animals either within a gnotobiotic isolator (Dec, Jan) or in hermetically sealed Tecniplast IsoCage P Bioexclusion cages on a rack
system (Mar, Oct), and allowed to acclimate for 3 days prior to colonization (Figure S1A). Mice were inoculated with a single dose of
the Bt RB-TnSeq mutant library at 106 - 108 CFU/200 mL. The inoculum was prepared by one of two methods: 1) frozen 2 mL aliquots
of the Bt RB-TnSeq library were thawed and gavaged directly into mice (Dec, Jan), or 2) a thawed 2 mL aliquot of the Bt RB-TnSeq
library was grown in 150 mL BHI-S medium overnight with 20 mg/mL erythromycin (16 h), and backdiluted to OD600 = 0.05 the next

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morning to allow for fresh cells to reach mid-log phase (3 h) by the time of inoculation (Mar, Oct). For each experiment, at least 3 cell
pellets of the inoculum were collected as Time = 0 references. Each mouse was colonized by oral gavage with 200 mL of the Bt trans-
poson library. Stool samples were collected daily (excluding weekends), up to 14 days post-colonization to assess longitudinal shifts
in mutant abundance. Mice were monitored and weighed daily. Genomic DNA was extracted using the DNeasy PowerSoil Kit.
The IsoCage P rack system facilitated sample collection while maintaining gnotobiotic conditions, and although the fresh and
frozen inocula exhibited nearly identical coverage of the Bt genome (Figure S1B) and harbored statistically indistinguishable levels
of diversity (Figures S1C and S1D), use of fresh rather than frozen inoculum substantially reduced bottleneck effects, allowing for low-
abundance mutants to reach the gut and persist at a much higher rate through at least D1 (Figure S1E), with diversity only beginning to
decrease at D4 (Figure 4A). Because of the bottleneck effect in the frozen inoculum cohorts, analysis of early time points in the func-
tional genetics experiments is restricted to experiments that used fresh inoculum (Mar, Oct). Results from later time points converged
across all four runs of the experiment, in spite of differences in protocol (Figures 1C and 4E).

Isolation of mutants from fecal matter

Mouse feces were collected and immediately homogenized in 500 mL 25% glycerol solution and stored at
80 C. Prior to isolation,
glycerol stocks were allowed to thaw on the benchtop for 10 min, centrifuged for 30 s at 2,000 RPM. On each 150 mm BHI-S plate,
100 mL of 10
3, 10
4, or 10
5 dilution of the glycerol stock was spread using 4.5 mm glass beads. The plates were incubated anaer-
obically at 37 C for two days. Individual colonies were picked into 1 mL 96-well plates containing 750 mL BHI-S in each well. After 16 h
of growth, glycerol was added to a final concentration of 20% and the isolates were stored. The isolate stocks were used as the
template in PCR amplifying the barcoded region of the mutants. The PCR products were sent for Sanger sequencing.

Host-associated evolution of spontaneous Bt mutants

Three female mice 8-12 wk-old C57Bl/6J GF mice were co-housed in the same gnotobiotic isolator and fed standard chow diet ad
libitum, and were then given a single dose of wildtype Bt VPI-5482 at 106 - 108 CFU/200 mL. One week post inoculation of Bt, the three
mice were separated into individual cages. A fecal sample was taken six weeks post inoculation. DNA was extracted from the fecal
pellets by the phenol-chloroform method, followed by ethanol precipitation, and sent for shotgun sequencing. Individual isolates from
each fecal pellet were cultured from the bulk material as outlined in Isolation of mutants from fecal matter. We assayed for isolates
with increased growth in VB-melibiose and selected one isolate from each mouse for MinION long-read sequencing.

Isolate genome sequencing, assembly, and polishing

To provide greater context for the delineation of the complex chromosomal rearrangements associated with the BT1872/BT1873 operon,
a long-read sequencing strategy was employed. The isolate genomes assessed were wildtype Bt, the strain used for the mouse exper-
iments, and three spontaneous mutant cultivars (MZ55, MZ58 and MZ65), which demonstrated enhanced growth rates in the presence of
melibiose, recovered from the feces of mice six weeks after initial inoculation. Total genomic HMW DNA was extracted by a standard
phenol chloroform protocol on overnight 25 mL BHIS broth cultures.63 DNA was resuspended in 0.1 mL 10 mM Tris-HCl, pH 8.5.
Slow pipetting, wide bore pipette tips and steps to minimize velocity gradients were implemented throughout to avoid further
shearing of DNA molecules. Libraries were prepared with the Rapid Barcoding Kit (SQK-RBK004) and the standard protocols
from Oxford Nanopore Technologies were used with the following modifications. DNA fragmentation was performed on 10 mg
DNA using 10 passes through a 22G needle in a 250 mL volume before purification using 0.5% Agencourt AMPure XP beads
(A63882, Beckman Coulter). Each elution step of the AMPureXP beads was performed using 10 mM Tris-Cl pH 8.5 instead of water,
at 37 C for 5 min. The gDNA inputs into library preparation ranged between 0.5 mg and 1.2 mg (Table S2), based on sample availability
in a standard 8.5 mL volume, with 1.5 mL Fragmentation mix added to each sample. Barcoded libraries were pooled so each sample
contributed an equal input mass (0.5 mg). Using MinKNOW v4.3.4, a single R9.4/FLO-MIN106 flow cell (Oxford Nanopore Technol-
ogies) sequenced the final prepared library with a starting voltage of
180 mV and a run time of 72 h. Guppy v5.0.11 and the sup
model were used for post-run basecalling, sample de-multiplexing and the conversion of raw FAST5 files to FASTQ files. For down-
stream analyses, we only used reads with a minimum quality score of 7. We assembled long-reads contigs with Flye.51 Additional
DNA extractions were carried out for every isolate using a standard phenol-chloroform extraction and send for short-read
sequencing. We then used the short-reads to polish the long-read assemblies using Pilon v1.23.52

Metabolite extraction from cecal material

Metabolites were extracted with the addition of extraction solvent (80% methanol spiked with internal standards and stored at

80 C, Table S4) to pre-weighed fecal/cecal samples at a ratio of 100 mg of material per mL of extraction solvent in beadruptor tubes
(Fisherbrand; 15-340-154). Samples were homogenized at 4 C on a Bead Mill 24 Homogenizer (Fisher; 15-340-163), set at 1.6 m/s
with 6 30-s cycles, 5 s off per cycle. Samples were then centrifuged at
10 C, 20,000 x g for 15 min and the supernatant was used for
subsequent metabolomic analysis.

Metabolite analysis using GC-EI-MS and methoxyamine and TMS derivatization

Metabolites were analyzed using gas chromatography-mass spectrometry (GCMS) with electron impact ionization. To a mass spec-
trometry autosampler vial (Microliter; 09–1200), 100 mL of metabolite extract was added and dried down completely under a nitrogen

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stream at 30 L/min (top) and 1 L/min (bottom) at 30 C (Biotage SPE Dry 96 Dual; 3579M). To dried samples, 50 mL of freshly prepared
20 mg/mL methoxyamine (Sigma; 226904) in pyridine (Sigma; 270970) was added and incubated in a thermomixer C (Eppendorf) for
90 min at 30 C and 1400 rpm. After samples are cooled to room temperature, 80 mL of derivatizing reagent (BSTFA +1% TMCS;
Sigma; B-023) and 70 mL of ethyl acetate (Sigma; 439169) were added and samples were incubated in a thermomixer at 70 C for
1 h and 1400 rpm. Samples were cooled to RT and 400 mL of Ethyl Acetate was added to dilute samples. Turbid samples were trans-
ferred to microcentrifuge tubes and centrifuged at 4 C, 20,000 x g for 15 min. Supernatants were then added to mass spec vials for
GCMS analysis. Samples were analyzed using a GC-MS (Agilent 7890A GC system, Agilent 5975C MS detector) operating in electron
impact ionization mode, using an HP-5MSUI column (30 m 3 0.25 mm, 0.25 mm; Agilent Technologies 19091S-433UI) and 1 mL in-
jection. Oven ramp parameters: 1 min hold at 60 C, 16 C per min up to 300 C with a 7 min hold at 300 C. Inlet temperature was 280 C
and transfer line was 300 C. Data analysis was performed using MassHunter Quantitative Analysis software (version B.10, Agilent
Technologies) and confirmed by comparison to authentic standards. Normalized peak areas were calculated by dividing raw
peak areas of targeted analytes by averaged raw peak areas of internal standards.

RT-qPCR
Total messenger RNA was isolated from colonic mucosal scrapings with TRIzol reagent according to the same protocol used for
cecal RNA isolation. Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics Corporation) was used to obtain cDNA.
Real-time qPCR was performed using iTaq Universal SYBR Green Supermix with CFX384 Real-Time System (Bio-Rad). Primers
are listed in Table S1.

ELISA
For cecal IgA ELISA, frozen cecal samples were resuspended in 1 mL of ELISA diluent per 100mg of cecal contents and homogenized
by bead beating for 1 min. ELISA was performed using goat polyclonal anti-IgA antibody (capture antibody) (Southern Biotech: 1040-
01) and goat anti-IgA antibody labeled with HRP (secondary antibody) (Bio-Rad: STAR137P). IgG ELISA was performed on frozen
serum samples using Invitrogen IgG (Total) Mouse Uncoated ELISA Kit.

Histological procedures
During sacrifice of mice colonized with WT Bt for transcriptomics experiments, distal colonic cross-sections were collected and
placed in cassettes. Tissues were fixed by immersion in Carnoy’s solution (60% ethanol, 30% chloroform, 10% acetic acid) for
3 h and were stored in 70% ethanol until tissue sectioning. When ready to process, samples were dehydrated by two successive
washes each in methanol for 35 min, ethanol for 30 min, and xylene for 25 min. Tissue samples within cassettes were then submerged
in melted paraffin at 68 C for 1hr, removed, and kept at room temperature until sectioning. Paraffin blocks were cut into 4-mm-thick
sections and deparaffinized for immunofluorescence.

Immunostaining and imaging

After deparaffinization and rehydration, slides were incubated in lysozyme solution at 37 C for 20 min, and then in antigen retrieval so-
lution (10 mM sodium citrate [pH 6.0]) at 90 C for 10 min. For mucus visualization, a polyclonal rabbit anti-mouse Muc2-specific antibody
(Santa Cruz Biotechnology) was diluted 1:100 in blocking buffer (Dako), applied to the slide, and incubated for 2 h in the dark at room
temperature. Slides were washed gently three times in TBS-T. The secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG, Invitrogen)
was diluted 1:100 in blocking buffer, applied to the slide, and incubated for 30 min in the dark at room temperature. Slides were again
washed gently three times in TBS-T, and were then stained with DAPI 10 mg/mL (Sigma), incubated for 1 min, and washed three times
in PBS. Slides were then dried, mounted with ProLong Gold Anti-Fade mounting medium (Invitrogen) and stored at room temperature in
the dark until imaging. Images were acquired on a Leica SP8 laser scanning confocal microscope with the LAS_X Leica software (Leica).
All samples were imaged with a 403 oil-immersion objective. Images were acquired at a frame size of 1024 x 1024 with 16-bit depth.

QUANTIFICATION AND STATISTICAL ANALYSIS

PCoA, differential expression analysis, and Gene Set Enrichment Analysis (GSEA) on transcriptomic data
The R53 package ’phyloseq’64 was used to calculate Bray-Curtis dissimilarity for all pairwise combinations of transcriptome samples,
which was then ordinated to create PCoA plots. PERMANOVA analysis using the R package ‘vegan’ was performed to evaluate sample
clustering by experimental day with significance criteria set at p < 0.05. Post-hoc pairwise PERMANOVA analyses using FDR method to
correct for multiple testing (‘pairwiseAdonis’) were then performed to evaluate significant pairwise differences in clustering (Table S3).
Gene calls were mapped to KEGG ortholog (Kofam) annotations in R. Differential expression analysis of each gene call was per-
formed by pairwise comparisons with ’deseq20 ,55 with the significance criteria log(FDR-adjusted p value) <
3, |log2(fold change)| > 2,
and max group mean >50 RPM. Metabolism for the Bt genome was estimated using the anvi’o v7.1 program ‘anvi-estimate-genome.’
Gene calls for each metabolic pathway found within the Bt genome were then transferred into a unique GSEA pathway query list in R
(‘fgsea’),57 and pathway enrichment was then calculated using the differential expression statistics calculated with ’deseq2’.55 Path-
ways were filtered for padj < 0.05 and the normalized enrichment scores (NES) was plotted (PRISMv9). Other custom gene lists were
created to calculate the enrichment of other gene sets including the polysaccharide utilization loci (PULs),20 capsular polysaccharide

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loci (CPSs),65 and genes associated with the Bt stringent response.16 Statistical results from these RNAseq analyses are presented in
Table S5.

Pipeline for measuring relative abundance and fitness scores of RB-Tn mutants
RB-TnSeq strain and gene fitness scores were calculated as described previously from strain-level count data (significance
threshold: t-statistic < -3s).14 For temporal abundance analyses of TnSeq mutants for each cohort, we first created a feature table
with raw counts of strain-level mutants across each sample. This table was filtered to remove strains with counts of 1, as these are
likely produced by sequencing error. Next, counts were normalized by the count of a synthetic spike-in barcode that was introduced
at 20 p.m. into each sample during PCR amplification of the barcodes. Samples where the spike-in represented >30% of total reads
were discarded. The synthetic spike-in barcode was subsequently removed as a feature from the table, and the resulting tables were
used for alpha diversity analyses via the R package ’vegan’.54 For all other relative abundance analyses, strains were assigned to
genes based on previous mapping by Liu et al.,3 as well as manual mapping performed for this experiment. Strains that had not
been mapped here or elsewhere were binned together as ‘‘non-mapping’’ strains, and strain-level counts were then summed for
each gene. For all subsequent analyses, we further filtered out genes that mapped to Bt plasmids in order to focus on chromosomal
gene fitness patterns. The R package ’phyloseq’ was used to calculate Bray-Curtis dissimilarity for all pairwise combinations of sam-
ples, which was then used to create PCoA plots.64 PERMANOVA analysis using the R package ‘vegan’ was performed to evaluate
sample clustering by experimental day with significance criteria set at p < 0.05 (Table S3). Finally, filtered count tables were adjusted
to relative abundance based on the total remaining counts, and used to track gene-level relative abundance over time. For linear
regression of initial vs. final relative abundance of gene mutants, genes with zero-counts were re-assigned a relative abundance
of 1x10
7 to perform log-transformation of the data.

Metabolomics statistics
As amino acid abundances (Figure 2E) were normalized within compounds and cannot be compared across compounds, for each
metabolite, we performed one-way ANOVA across all time points, with post-hoc follow-up tests and FDR correction (Table S4).
Carbohydrate metabolite abundances (Figures 5D, S3C and S3D) were compared within compounds across time points using
one-way ANOVA with post-hoc follow-up tests and FDR correction (Table S6).

Bacterial growth rate statistics

Growth rates of different Tn mutant isolates (Figures 5A and S3B) were compared by one-way ANOVA with post-hoc follow-up tests
and FDR correction (Table S6).

Genome mapping and coverage visualization

We used anvi’o v7.156 and the pangenomic workflow to compute and visualize genomic coverage for each isolate genome from the
evolution experiments. Briefly, the workflow uses (1) Prodigal v2.6.358 to identify open-reading frames (ORFs), (2) ’anvi-run-hmm’ to
identify single copy core genes from bacteria (n = 71)66 and ribosomal RNAs (n = 12, modified from1) using HMMER v3.3,67 (3) ’anvi-
run-ncbi-cogs’ and ’anvi-run-kegg-kofams’ to annotate ORFs with the NCBI’s Clusters of Orthologous Groups (COGs),68 and the
KOfam HMM database of KEGG orthologs (KOs)69,70 respectively. We used bowtie2 v2.3.5.159 to recruit short-reads to the contigs,
and samtools v1.1160 to convert SAM files to BAM files. We profiled the resulting BAM files with ’anvi-profile’ and used the program
’anvi-merge’ to combine all single profiles into a merged profile for downstream visualization. We used ’anvi-get-split-coverages’ and
’anvi-script-visualize-split-coverages’ to generate coverage plots. We used ’anvi-export-gene-calls’ and gggenes v0.4.1 to visualize
the genomic context around BT1871.

BT1871 copy number

We used blast71 to compute the number of long-reads with two copies of the BT1871 locus in the MZ65 isolate. We extracted the gene
sequence (1989 bp) from the initial Bt genome (AMD595) using anvi’o interactive interface and used blastn v2.5.0 to blast the long-
reads from MZ65. Blast hits with an alignment length >180% of the gene length were flagged as "two copies" and hits with alignment
length between 80% and 105% were flagged as "one copy". To visualize long-reads with two copies of BT1871, we used minimap2
v2.1761 to map the MZ65 long-reads to the MZ65 genome, which had two copies of the BT1871 region. We used samtools v1.11 to
extract the reads with two copies as identified above and used IGV v2.11.162 to visualize the mapping and generate a figure.

Image quantification
For each mouse, a single colonic cross-section was used to generate 6 representative images. Images were masked to split the DAPI
signal into an epithelial channel (blue) and a luminal channel (red). The MUC2 channel was green. To assess proximity of Bt to the
epithelium, at 10 evenly-spaced points along the epithelium in each frame, a perpendicular line was drawn until it intersected the
nearest bacterial cell, and this distance was measured. These measurements were averaged across all frames for each mouse.
Average measurements of n = 3–4 mice per time point were used to compare Bt epithelial proximity across experimental days by
performing one-way ANOVA and then post-hoc follow-up tests with FDR correction to compare all time points to D1. Differences
were considered significant at q < 0.05.

Cell Reports 42, 113009, August 29, 2023 21

Cell Reports, Volume 42

Supplemental information

Dynamic genetic adaptation

of Bacteroides thetaiotaomicron
during murine gut colonization
Megan S. Kennedy, Manjing Zhang, Orlando DeLeon, Jacie Bissell, Florian Trigodet, Karen
Lolans, Sara Temelkova, Katherine T. Carroll, Aretha Fiebig, Adam Deutschbauer, Ashley
M. Sidebottom, Joash Lake, Chris Henry, Phoebe A. Rice, Joy Bergelson, and Eugene B.
Chang
 A B
Dec. gavage with

Mean Insertions per 20kb

Jan. thawed solution

frozen
stock
gnotobiotic isolator

gavage with
Mar. fresh solution
Oct.

gnotobiotic IsoCage P
grow overnight
frozen + backdilute
stock
Chromosome Position (kb)

C n.s.
E
log(D1 relative abundance)

D n.s.

log(D0 relative abundance)

Fig. S1: Comparison of functional genetics experimental protocols and outcomes. A) Schematic
representation of protocol differences across experimental cohorts. In Dec. and Jan. cohorts, mice were
housed in cages of 2-3 animals within gnotobiotic isolators and gavaged with thawed stocks; in Mar. and
Oct. cohorts, mice were housed in gnotobiotic cages of 2-3 animals on an IsoCage P Bioexclusion rack
system and gavaged with stocks that had been grown overnight in fresh media. B) Mean number of
unique mutant strains with RB-Tn insertions identified per 20kb region in inoculum samples from each
experimental cohort. C) Rarefied strain richness is not significantly different across fresh and frozen
inocula (two-sided Wilcoxon rank sum test, p = 0.09, n= 8–13 per group). Samples were rarefied due to
uneven sequencing depth across experimental cohorts. D) Shannon diversity is not significantly different
across fresh and frozen inocula (two-sided Wilcoxon rank sum test, p = 0.1774). E) Comparison of
inoculum (D0) versus D1 relative abundance of each RB-Tn gene mutant. Dashed line represents 1:1, red
line and equation represent linear regression best-fit line. Cohorts with frozen inoculum (Dec, Jan)
experienced greater loss of mutants with low D0 relative abundance (< 0.0001) compared to cohorts with
fresh inoculum (Mar, Oct).
Fig. S2: Pathway map of reactions related to amino acid biosynthesis. Red arrows represent specific
genes whose transcript is significantly enriched on D0.5/1 relative to D2/4 (logFDR < -3, |log2FC| > 2,
and base mean > 50 RPM), and red highlight represents pathways that were overall enriched on D0.5/1
relative to D2/4 (padj < 0.05). No genes or pathways on this map were relatively more expressed at
D2/D4 than D0.5/D1. Genes whose transcript level did not differ significantly between D0.5/1 and D2/4
are colored in black. Grey dashed arrows represent reactions for which the associated gene is unknown in
the Bt genome. Gold stars represent gene disruptions that were depleted in the RB-Tn assay, as in Fig. 2C.
See also Table S6.
Fig. S3: Bt adapts to the GF gut specifically by increasing efficiency of metabolizing α-1,6 bonded
sugars. A) Structure of the trisaccharide raffinose. B) The log phase doubling times of Tn_BT3133 and
Tn_BT1872 were measured in Varel-Bryant medium with 20 mM raffinose, melibiose, sucrose,
galactose, or glucose as the sole carbon substrate. Abundance of C) melibiose or D) sucrose in the
standard chow fed to GF mice, within GF ceca before colonization, or 1, 7, or 14 days post-colonization.
Fig. S4: Bt does not elicit a strong temporal host response. A) Cecal IgA and B) serum IgG levels from
mice sacrificed at different timepoints after colonization (n=3-4/timepoint). C) Cytokine expression
relative to gapdh in colonic mucosal scrapings at different timepoints after colonization (n=4-
6/timepoint).
 Fig. S5: Capsular polysaccharide biosynthesis operons are significantly associated with different
stages of colonization. (A – D) Gene set enrichment analysis (GSEA) using transcriptomics data for
capsular polysaccharide biosynthesis operons, comparing sequential timepoints. Only statistically
significant (adj. p < 0.05) scores are shown. (E) Measurement of the adjusted t-statistic (12) in the RB-Tn
assay on all days where fitness score was measurable (D1-3) shows that gene insertions in CPS4
consistently result in significant declines in mutant fitness for all genes within the CPS4 locus except for
BT1338. See also Table S5.

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